Chemicals of sequenced units, such as proteins which are composed of sequences of amino acids may be produced by solid phase chemistry wherein chemical units attached to a solid support are reacted with chemicals that attach other selected units to the units attached to the solid support to form unit chain compounds. Such two unit compounds may then be treated with the same or other chemicals repeatedly to form any predetermined number of units of sequenced compounds.
By providing photolabile protecting groups to the exposed units and sequentially unmasking certain areas of such units by light irradiation and exposing such unprotected areas to a reactive chemical disposed to attach a desired unit to the exposed units it is possible to create two unit chains in a confined area of the support. By repeating such steps in the same or other areas of the support and repeating varying chemical treatments as set forth above it may be readily seen that numerous differing chemical chain compounds may be effected in a confined space on a single support.
This technology is of particular significance in the biological production of antibodies with sequences of amino acid units that will exhibit a high level of binding and specificity for a protein. The binding of an antibody to a specific antigen is critically important in biological systems, since the specificity and strength of the antibody-antigen interaction is crucial to the operation of the immune system. The specificity of an antibody for a protein has been traced to highly variable segments in the antibody chain generally consisting of 6 to 10 amino acid sequences. It has been found that antibody segments containing one highly variable sequence, called a domain, exhibit high levels of binding and specificity for a protein ("Single-Domain Antibodies Promise New Chemical, Medical Applications" C&E News, Nov. 20, 1989).
The determination of the set of polypeptides that bind specifically to any given protein would be of obvious use in a variety of therapeutic and diagnostic treatments. Brute force determination of the sequences has been thought of as all but impossible. There are 20 common amino acids, so the number of possible 6 member polypeptides is 20.sup.6, or 64 million.
A recent development has shown promise in determining these sequences, through the synthesis of large numbers of peptides bound to a solid support. This method combines techniques of solid phase peptide synthesis, photolabile protecting groups, and photolithography to achieve light directed, spatially addressable parallel chemical synthesis. The method works as follows. A solid support (typically a glass microscope slide) is reacted with aminopropyltrie-thoxysilane to form amine groups on its surface. These groups are then reacted with an amino acid whose amine function is protected by a photolabile protecting group. The surface is then masked, for example, the surface is everywhere covered except for a rectangular strip exposing 5% of the exposed surface. The surface is then irradiated with UV light, activating only 5% of the exposed surface. The surface is then reacted with an amino acid whose amine function is also protected by a photolabile group; only the activated portion of the surface reacts with the amino acid. The masking, irradiation, and reaction step is repeated, with a different area exposed and a different amino acid reacted with the surface. After 20 steps, 20 parallel strips exist on the surface, each strip containing a different amino acid bound to the surface.
This process is then repeated, but with masks rotated to a position perpendicular to those used in the first masking sequence. At the end of the process all possible two member polypeptides have been synthesized on the solid support. Each polypeptide combination is located in squares formed from the intersection of the strips. The above procedure may be repeated to achieve any number of differing amino chain combinations in such intersecting squares.
This method has been used to prepare 1,024 different polypeptides on a square centimeter of surface. Utilizing the method of the present invention the potential exists for the synthesis of millions of polypeptides bound on a solid support. This method has also been used to synthesize different oligonucleotides on a solid support.